The search for new industrial enzymes and more specifically secreted enzymes is presently reliant on the availability of simple primary functional assays. Typically the substrate is used in the growth medium for the screening of microorganisms and degradation of the substrate may be recognized by a physical change in the substrate (colour change, halo formation around a colony, fluorescence etc.). Many proteins exist for which there is no simple functional assay and these may have potential application as industrial enzymes.
Enzymes which are secreted are highly interesting for use in industrial applications. A positive selection screening system which selects only clones encoding secreted enzymes is thus very desirable. Signal trapping is a method to identify genes containing a signal peptide using a translational fusion to an extracellular reporter gene lacking its own signal. This has been reported in the literature for the purpose of identifying new signal sequences (Manoil & Beckwith 1985, TnphoA: A transposon probe for protein export signals. Proc. Natl. Acad. Sci USA 82:8129–8133; Smith, H. et al., 1987, Construction and use of signal sequence selection vectors in Escherichia coli and Bacillus subtilis. J. Bact. 169:3321–3328), also the use of such for defining clearly the specific elements within signal peptides which are required for optimal function (Smith, H. et al, 1988. Characterisation of signal-sequence-coding regions selected from the Bacillus subtilis chromosome. Gene. 70:351–361).
A number of publications describe cloning vector reporter systems where genomic or cDNA libraries are constructed in a screening vector containing a signal-less reporter gene. When a cDNA or genomic fragment lacking a translational stop site is cloned upstream of the reporter gene in a translational fusion, a resulting protein-reporter gene fusion product is formed. If the cDNA or genomic fragment cloned contains a signal peptide, the fusion protein is secreted to the outside of the cell. Secretion can be detected by growth on selective media as in the use of invertase in Saccharomyces cerevisiae or in the use of e.g. β-lactamase in Escherichia coli. These publications are not concerned with methods for screening previously established gene libraries.
The number of clones to be investigated in the library is dramatically reduced by the screening to those containing a signal peptide, however a resulting clone may only contain an incomplete gene which may or may not include the minimum DNA information needed to encode the enzymatic activity originally associated with the secretion signal sequence isolated.